The Maintenance And Regulatory Mechanisms Of Leptin A In Energy Homeostasis Of Nile Tilapia(Oreochromis Niloticus)

Leptin is known to inhibit appetite and promote energy metabolism throughout the vertebrates.In mammals,hyperleptinemic obesity with inability of exogenous leptin to reduce feeding and body weight is referred to as leptin resistance.Often,leptin resistance is commonly observed in diet-induced obesity(DIO).However,the roles of leptin in the energy homeostasis in DIO animals with leptin resistance remain unclear.Given that teleosts are the first true vertebrates,the physiology of teleostean leptin should be more original and best represented.Nevertheless,knowledge about the mechanisms of teleostean leptin in energy homeostasis is currently limited.In addition,little is known about leptin resistance in teleost and the roles of leptin in energy metabolism under over-nutritional statuses are still unknown.In this study,Nile tilapia(Oreochromis niloticus)was chosen as a model fish to study the role of leptin in energy homeostasis.Molecular cloning,quantitative real time PCR,Western Blot,immunohistochemistry(IHC),histology analysis,transcriptomic analysis,and other approaches were used in the following studies: first,we cloned the Leptin A in Nile tilapia,determined the tissue distribution of Leptin A,and obtained the recombinant Leptin A.Secondly,we determined the roles of Leptin A in appetite and energy metabolic regulation with different nutritional statuses and recombinant Leptin A treatment.Finally,we generated two obese fish models by feeding high-carbohydrate(HCD)or high-fat diet(HFD)for eight weeks and analyzed comprehensively the metabolic characteristics of two obese fish models.On this basis,we determined the expression of Leptin A,the leptin signal status and leptin resistance in these two obese fish models.Furthermore,we assessed the roles of Leptin A in energy metabolic regulation under obese statuses.In addition,we also cloned the SOCS-3 which plays a critical role in leptin signaling,as well as SOCS-1 and SOCS-2,and determined the factors that affect the transcriptional expression of SOCS-1,2 and 3.1.Gene cloning,tissue distribution and prokaryotic expression of Leptin AThe first teleostean leptin was cloned more than a decade after the first mammalian one.As the species of fish are numerous,cloning leptin gene in different fish species represents the first and basic approach before further aspects are studied.In this study,we cloned Leptin A in Nile tilapia and analyzed the homology of the amino acid sequence and the three-dimensional structure of the protein.The full-length cDNA of Leptin A was 486 bp,encoding a protein of 161 amino acid(aa)which contained a signal peptide in the first 20 aa.The homology of the leptin aa sequences among different species were very low,and Nile tilapia and human only shared 15.3% identity of aa sequences,but the three-dimensional structure of the protein was conserved.Previous studies indicated that fish leptin mRNA was mainly expressed in liver,but these results were not verified at the protein level.Here,we found that Leptin A was expressed in all 11 tissues.Consistent with the findings of many studies in fish,the highest Leptin A mRNA expression levels were observed in the liver.Surprisingly,further IHC analysis with a Leptin A-specific antibody indicated that Leptin A is expressed in liver and also in subcutaneous adipose tissue(SCAT).These results indicate that SCAT is another site with the high expression of Leptin A.In addition,we constructed a prokaryotic expression system and obtained the recombinant Leptin A for following experimental study.2.The regulatory role of Leptin A on appetite and metabolism and its mechanismLeptin plays a critical role in appetite and metabolic regulation,and the regulatory mechanism has been clarified in mammals.However,studies on the regulation of appetite and energy metabolism in fish leptin are limited and the mechanism involved is unclear.In the present study,we determined the dynamics expression of Leptin A and LepR in Nile tilapia from satiation to starvation and further assessed the effect of i.p.injected recombinant Leptin A on appetite and metabolism.The results showed that the expression of Leptin A in the liver of Nile tilapia increased from satiation to starvation,while decreased in SCAT.Moreover,the expression of LepR in different tissues showed different peaks depending on time.The LepR in the fed Nile tilapia decreased after 12 h in the brain,12 h in the liver,36 h in the subcutaneous fat and 72 h in the visceral adipose tissue(VAT).Many studies have indicated that the expression of leptin decrease due to hunger stress in mammals,while increase in fish.Therefore,the different expressions of Leptin A between liver and SCAT under starvation may maintain the regulation of metabolism and appetite,respectively.The highest expression of LepR in different tissues at various time points suggests that leptin may has a sequence of energy metabolic regulation in different tissues.The results of i.p.injected recombinant Leptin A showed that it activates the downstream signaling pathway by binding to the LepR in the hypothalamus and inhibits the expression of neuropeptide Y(NPY)to inhibit the appetite.Moreover,Leptin A increased the expression of adipose triglyceride lipase(ATGL),apolipoprotein E(apoE)and glucose transporter 2(Glut2)to accelerate lipolysis and glycogenolysis.Overall,our results indicated that Leptin A has the functions of inhibiting appetite and activating nutrients catabolism in Nile tilapia.3.Exploration of leptin resistance in teleosts and the roles of leptin in energy metabolism under obese statusLeptin resistance is commonly observed in obese mammals and the most commonly used model for leptin resistance research is high-fat diet induced obesity(DIO)in mice or rats.In fish,knowledge about obese fish model is limited.Therefore,there is no evidence to confirm the existence of leptin resistance in teleosts and the roles of leptin on energy metabolism under obese status is also unclear.In this study,we generated two types of DIO Nile tilapia with a high-carbohydrate diet(HCD)or a high-fat diet(HFD).Metabolic characteristics analysis showed that more muscle glycogen and SCAT were stored in HCD-fed fish and more VAT was stored in HFD-fed fish.Insulin resistance occurred in HFD-fed fish but not HCD-fed fish.The two DIO fish models had distinct mechanisms of fat deposition as suggested by the decreased expression of PPAR? in HCD-fed fish and increased expression of PPAR? in HFD-fed fish.Exploration of leptin resistance found that both two DIO models showed hyperleptinemia,but the main sites of Leptin A synthesis in HCD-and HFD-DIO fish were SCAT and liver,respectively.We observed low p-STAT3 and high expression of SOCS-3 in hypothalamus demonstrating impaired leptin signaling.Furthermore,the exogenous Leptin A lost actions of appetite and body weight regulation.These results confirmed the existence of leptin resistance in both DIO fish models.Most importantly,in HCD-fed fish,exogenous Leptin A did not affect carbohydrate metabolism,but maintained its action by selectively regulating lipid metabolism through decreasing plasma triglyceride(TG),increasing plasma nonesterified fatty acid(NEFA).Conversely,in HFD-fed fish,the exogenous Leptin A could not affect lipid metabolism,but still selectively regulated carbohydrate metabolism by increasing glucose plasma concentration.The results indicated that Leptin-resistant DIO Nile tilapia selectively retain leptin action in lipid or glucose metabolism under different over-nutrition statuses.Previous study demonstrated that the fat storage is required for a blockade of Leptin signaling.In addition,fat storage is for survival in food shortage period.One week fasting completely recovered leptin actions in lipid and glucose metabolic regulations in the two DIO tilapia models.Therefore,the selectively regulated nutrients metabolism in DIO fish leptin may be an adaptive strategy for storing efficiently fat deposits.4.Molecular characterization and transcriptional analysis of SOCS,a key feedback regulator of leptin signalingSuppressor of cytokine signaling 3(SOCS-3)is a key negative feedback regulator of leptin signaling and is closely associated with the occurrence of leptin resistance.However,it is also involved in other cytokines and hormone signaling regulations.However,the transcription of SOCS-3 is affected by many other factors.To determine the factors that affect the transcription of SOCS-1,2 and 3 in Nile tilapia,the cDNAs of SOCS-1,2 and 3 in Nile tilapia were cloned.Lipopolysaccharide(LPS)stimulation,different nutrition treatments,insulin injection,bacterial stimulation,insulin-like growth factor 1(IGF-1)injection and dual fluorescein report assay were performed to analyze systematically the effects of SOCS-1,2 and 3 transcription.The homology of the SOCSs aa sequences among different species were very conserved and they all belong to the type II subfamily by phylogenetic analysis.Tissue distribution analysis showed that SOCS-1 and SOCS-3 are mainly expressed in gills and other immune related tissues,while SOCS-2 is mainly expressed in the liver.The results of transcriptional analysis showed that,LPS up-regulated SOCS-3 transcription,while down-regulated SOCS-2 transcription both in vivo and in vitro.The transcription of SOCS-2 and IGF-1 has a positive correlation under different nutritional statuses.Insulin injection up-regulated SOCS-3 transcription and down-regulated SOCS-2 and IGF-1 transcriptions.Bacterial stimulation up-regulated the transcription of SOCS-1 and SOCS-3,while down-regulated the transcription of SOCS-2 and IGF-1.Further experiments with rhIGF-1 injection and double luciferase report showed that IGF-1 promoted the transcription of SOCS-2.These results suggest that LPS and bacteria caused immune response and insulin can significantly promote the transcription of SOCS-3.The functions of SOCS-1 may be more inclined to regulation in the immune response while SOCS-2 may be involved in the regulation of endocrine factor signaling.Finally,IGF-1 can promote the expression of SOCS-2 transcripts.